Candida albicans two-component hybrid kinase gene, CaNik1, and use thereof

ABSTRACT

A  Candida albicans  gene, CaNik1, is involved in phenotypic switching which is significant because of a direct correlation between the switching and the level of virulence of the organism. A method of screening for anti-fungal pharmaceutical candidates entails bringing a test substance into contact with cells containing a CaNik1 gene or a variant thereof and then monitoring the effect, if any, on the level of expression of the gene.

This application claims benefit of provisional application 60/048,914filed Jun. 6, 1997.

This application describes microorganisms that have been deposited, inaccordance with the Budapest Treaty, under ATCC Patent DepositDesignation: PTA-4456, with the following Deposit IdentificationReference: Bacteriophage lambda EMBL3: Ca lambda 15.1.

BACKGROUND OF THE INVENTION

Candida is an opportunistic yeast that lives in the mouth, throat,intestines, and genitourinary tract of most humans. In a healthy humanbody, the population of Candida is kept in check by the immune systemand by a competitive balance with other microorganisms. But when thebody's immune system is compromised, as in AIDS patients and in patientsundergoing immunosuppressive therapy, Candida will grow uncontrolled,leading to systemic infection called “Candida mycosis.” If leftuntreated, such systemic infections frequently lead to the death of thepatients.

Candida albicans is a species of particular interest to scientists anddoctors because 90% of all cases of Candida mycosis are caused by thisspecies.

At present, the therapy principally available for invasive infections isbased on relatively few antimycotics, such as amphotericin B andflucytosine, or the azole derivatives fluconazole and itraconazole.These antimycotics cause serious side effects, such as renalinsufficiency, hypocalcaemia and anaemia, as well as unpleasantconstitutional symptoms such as fever, shivering and low blood pressure.Amphotericin B is toxic to the kidneys, for example, and yet thepharmaceutical is therapeutic only if administered at dose levels nearto being toxic. A discussion of the pharmaceuticals used for treatmentand their corresponding side effects can be found, for example, in Boyd,et al., BASIC MEDICAL MICROBIOLOGY (2d ed.), Little, Brown and Company,(1981).

Given the deficiencies of conventional therapies against Candida, a needexists for developing pharmaceuticals that are effective in this regardand also safe to use. One step in the development of suchpharmaceuticals requires a method for screening compounds in order toidentify pharmaceutical candidates.

SUMMARY OF THE INVENTION

It therefore is an object of the present invention to provide anisolated polynucleotide sequence coding for a protein that is linked tophenotypic switching in Candida albicans.

It is a further object of the invention to provide a method forscreening compounds to identify pharmaceutical candidates foreffectively inhibiting the pathogenicity of C. albicans.

In accomplishing these and other objects, there has been provided,according to one aspect of the present invention, an isolatedpolynucleotide that codes for such a protein and that hybridizes, understringent conditions, to the polynucleotide sequence of SEQ ID NO:1,shown below in FIG. 1. In a preferred embodiment, the polynucleotide hasthe sequence of SEQ ID NO:3 (FIG. 2). In another preferred embodiment,the protein displays a kinase activity.

In accordance with another aspect of the present invention, a method isprovided for screening compounds to identify pharmaceutical candidates.The inventive method comprises the steps of (A) providing a plurality ofcells from yeast species that exhibit phenotypic switching, at leastsome of which contain (i) a polynucleotide coding for a CaNIK1 proteinand (ii) a promoter that is operably linked to the polynucleotide, suchthat the plurality of cells produces the protein; then (B) bringing theplurality into contact with a test substance; and (C) assessing whateffect, if any, the test substance has on the expression of the DNAsegment. Assessment step (C) can comprise, for example, of monitoringthe level either of the protein or the corresponding mRNA transcriptproduced by the plurality of cells. In another embodiment, step (C)comprises monitoring the level of kinase activity, within the plurality,that typifies the protein.

In yet another embodiment of the present invention, a promoter isoperably linked to a reporter gene. In this context, step (C) comprisesmonitoring the level of transcription of the reporter gene, aftercontact between the plurality of cells and the test substance.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, only indicate preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show the nucleotide sequence (SEQ ID NO:11) (top row) of thePCR product encoding the region spanning the H1 and D domains and thededuced amino acid sequence of the CaNIK1 protein (SEQ ID NO:2) (bottomrow). The amino acid residues of functional domains are underlined. Thethree degenerate primers used to isolate the PCR products are shown asSlb1, Slb2, and Slb3.

FIGS. 2A-E show the nucleotide sequence(SEQ ID NO:3) (top row) of thegene CaNik1 and the deduced primary amino acid sequence of the CaNIK1protein(SEQ ID NO:11) (bottom row). The beginning of each unique repeatis represented within the rectangle. The potential amino acid residuesof different functional domains are underlined.

FIG. 3 is a schematic representation of the anatomy of two alleles intwo strains of C. albicans according to the present invention. All thefunctional domains are shown as white bold letters inside eachrectangle. A few of the unique restriction enzyme sites are shown at thetop of the rectangle. The start of the protein coding region is shown asATG. WO-1 and CAI8 are the two strains analyzed in this invention. H1and H2 are two identical alleles of the strain WO-1. H1-L and H2-Srepresent large and small alleles respectively in strain CAI8. The fivehatched rectangular units in each allele represent repeat unitsdescribed in this invention. The gray rectangular area encompassingXhoI-PstI in H2-S represents the region containing a deletion ofapproximately one repeat unit length.

FIG. 4 illustrates the deletion strategy used to generate a homozygousdeletion mutant, HH80, in strain CAI8. The region spanning AflII-XhoIwas deleted and substituted by a hisG-Urablaster cassette in the plasmidpUNIK12.1 to create pCNH35 (FIG. 4c). Plasmid pUNIK12.1 (FIG. 4b) wasderived by subcloning a PCR fragment using a pair of primers Slb8 andSlb7R and subcloning into pGEM-T easy plasmid vector. λSA15.1 representthe lambda clone identified in a screen that contain the genomicfragment encompassing the entire CaNik1 gene and the flanking DNAsequence.

FIG. 5 shows the deletion strategy used to generate the homozygousdeletion mutant in Red 3/6, an ade2⁻ derivative of strain WO-1. Thedeletion cassette pABX12 (FIG. 5b) was generated by deletion of all thefunctional domains except H2 and substitution with the ADE2 gene as anauxotrophic marker in pUNIK12.1 (FIG. 5c). FIG. 4 provides a descriptionof λSA15.1.

Table 1 summarizes the effects of the CaNik1 deletion in HH80 on growthin a variety of solution and conditions, high frequency phenotypicswitching, and dimorphism.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Candida albicans is capable of differentiating in a reversible fashionbetween a bud and a hyphal growth form. Each strain of C. albicans canalso undergo high frequency phenotypic switching between a limitednumber of general phenotypes that differ in a variety of traitsincluding putative virulence factors. The frequencies of both of thesedevelopmental programs are influenced by environmental conditions. Forexample, pH and temperature influence the transition between bud andhypha while temperature, UV, white blood cell metabolites and colonyaging affect the frequency of high frequency phenotypic switching. Themorphological changes made by C. albicans in response to environmentalcues indicates that the organism uses a sensory mechanism to registerand assess environmental alterations.

Autophosphorylating histidine kinases, also known as “two-componentresponse regulators,” have been found, in lower eukayotes such as fungiand slime molds, to play a pivotal role in relaying variousenvironmental signals into the cell for inducing appropriate responsesand in providing these organisms with the capacity to respond rapidly toan environmental perturbation. Two-component signal transducers allcontain a sensory kinase, which autophosphorylates a histidine residuein response to an environmental cue, and a response regulator, whichthen is phosphorylated and, through a resultant conformational change,effects a signal that is transduced either directly to a molecularcomplex, as in the case of the bacterial CheY and the flagellar motor,or down a signal transduction pathway, as in the case of SLN1. Theseproteins have been shown to be involved in regulating morphogenesis anddevelopment in various prokaryotes and eukaryotes.

That two-component response regulators have been identified in otheryeast species suggests that the two-component response regulators mayalso play a role in the developmental programs of C. albicans. Thepresent invention relates to such a two-component response regulator,the hybrid kinase CaNIK1 from Candida albicans. A link between the geneencoding CaNik1 and the processes of phenotypic switching that includesthe differential expression of pathogenic genes is evidenced by workwith a CaNik1-deletion strain of C. albicans. See examples 3 and 5.Thus, CaNik1 is know to be involved in phenotypic switching.

Phenotypic switching is thought to be linked to the virulentcharacteristics of yeast. Candida albicans switches phenotypes withregard to its environment in order to maximize pathogenesis according tothe demands of the particular environment. For example, in the WO-1strain of Candida albicans, studies have shown that the yeast is morevirulent in its opaque phenotype when located on the skin. When WO-1 isin the white phenotype, however, it is more pathogenic in systemicinfections. A description of the relationship between the phenotypicswitching and the pathogenic characteristics of Candida albicans can befound in Soll, “Switching and Gene Regulation in Candida albicans,” inSOCIETY FOR GENERAL MICROBIOLOGY SYMPOSIUM 50 (1992). This relationshipbetween phenotypic switching and pathogenicity can be exploitedeffectively, in a bioassay, for the purpose of discoveringpharmaceutical candidates against Candida albicans.

1. Definitions

In this description, “isolated DNA” is a fragment of DNA that is notintegrated in the genomic DNA of an organism. For example, the CaNik1gene is a DNA fragment that has been isolated from the genomic DNA of C.albicans.

As used herein, “protein” refers to a polymer of amino acid residues.The terms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. Exemplary modifications aredescribed in most basic texts, such as PROTEINS—STRUCTURE AND MOLECULARPROPERTIES (2d ed.), T. E. Creighton, W. H. Freeman and Company, NewYork (1993).

As used herein, “selectively hybridizes” includes reference tohybridization, under stringent hybridization conditions, of a nucleicacid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences typically have about at least 80% sequenceidentity, preferably 90% sequence identity, and most preferably 100%sequence identity (i.e., complementary) with each other.

The terms “stringent conditions” or stringent hybridization conditionsincludes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, preferably less than 500 nucleotides in length.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth & Wahl, Anal. Biochem. 138: 267-84 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. But severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point(T_(m)); moderately stringent conditions can utilize a hybridizationand/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal meltingpoint (T_(m)); low stringency conditions can utilize a hybridizationand/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermalmelting point (T_(m)). Using the equation, hybridization and washcompositions, and desired T_(m), those of ordinary skill will understandthat variations in the stringency of hybridization and/or wash solutionsare inherently described. If the desired degree of mismatching resultsin a T_(m) of less than 45° C. (aqueous solution) or 32° C. (formamidesolution) it is preferred to increase the SSC concentration so that ahigher temperature can be used. An extensive guide to the hybridizationof nucleic acids is found in Tijssen, LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY—HYBRIDIZATION WITH NUCLEIC ACIDPROBES, Part I, Chapter 2 “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” Elsevier, New York (1993);and in Chapter 2 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, GreenePublishing and Wiley-Interscience, New York (1995) (hereafter “Ausubelet al.”).

Two nucleic acid molecules are considered to have a “substantialsequence similarity” if their nucleotide sequences share a similarity ofat least 50%. Sequence similarity determinations can be performed, forexample, using the FASTA program (Genetics Computer Group; Madison,Wis.). Alternatively, sequence similarity determinations can beperformed using BLASTP (Basic Local Alignment Search Tool) of theExperimental GENIFO(R) BLAST Network Service. See Altschul et al.,“Sequence Similarity Searches, Multiple Sequence Alignments, andMolecular Tree Building,” in METHODS IN PLANT MOLECULAR BIOLOGY ANDBIOTECHNOLOGY, Glick et al. (eds.), pages 251-267 (CRC Press, 1993).

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription.Tissue-specific, tissue-preferred, cell type-specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is one that is active under most environmentalconditions.

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

As used herein, “expression” refers to the biosynthesis of a geneproduct. For example, in the case of a structural gene, expressioninvolves transcription of the structural gene into mRNA and thetranslation of mRNA into one or more polypeptides.

As used herein, “expression vector” is a polynucleotide moleculecomprising a gene that is expressed in a host cell. Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene is said to be “operablylinked” to the regulatory elements.

2. Isolating a Candida albicans Polynucleotide Segment Encoding CaNik1Protein

An endogenous polynucleotide sequence from Candida albicans whichencodes for the CaNIK1 protein was isolated using a polynucleotide probederived from PCR amplification. See Example 1. Hybridization of theprobe against a genomic library resulted in the determination of thefull length polynucleotide sequence encoding the CaNIK1 protein. SeeExample 2. The full polynucleotide sequence encapsulating the CaNik1gene is provided in FIG. 2.

3. Nucleic Acids

The present invention provides, inter alia, isolated nucleic acids ofRNA, DNA, and analogs and/or chimeras thereof, comprising apolynucleotide encoding a CaNIK1 protein or a polynucleotide probe whichhybridizes to a polynucleotide encoding CaNIK1 protein. In this regard,the invention provides the nucleotide sequences of FIGS. 1 and 2. Inaddition, the present invention also provides other sequences asdescribed below.

a. Polynucleotides Encoding A CaNIK1 Polypeptide or ConservativelyModified or Polymorphic Variants Thereof

As indicated above, the present invention provides isolated heterologousnucleic acids comprising a polynucleotide, wherein the polynucleotideencodes a CaNIK1 protein, disclosed herein in FIG. 2, or conservativelymodified or polymorphic variants thereof. Those of skill in the art willrecognize that the degeneracy of the genetic code allows for a pluralityof polynucleotides to encode for the identical amino acid sequence. Such“silent variations” can be used, for example, to selectively hybridizeand detect allelic variants of polynucleotides of the present invention.Accordingly, the present invention includes polynucleotides that aresilent variations of the polynucleotides of FIG. 2. The presentinvention further provides isolated nucleic acids comprisingpolynucleotides encoding conservatively modified variants of CaNIK1encoded by the sequences in FIG. 2. Conservatively modified variants canbe used to generate or select antibodies immunoreactive to thenon-variant polypeptide. Additionally, the present invention furtherprovides isolated nucleic acids comprising polynucleotides encoding oneor more polymorphic (allelic) variants of polypeptides/polynucleotides.

b. Polynucleotides That Selectively Hybridize

The present invention also provides isolated nucleic acids comprisingpolynucleotides, wherein the polynucleotides selectively hybridize,under selective hybridization conditions, to a polynucleotide asdiscussed above. In this regard, the present invention encompassespolynucleotides that selectively hybridize, under selective conditions,to a polynucleotide as discussed above, excluding the polynucleotide ofFIG. 2. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising thepolynucleotides described above. For example, polynucleotides of thepresent invention can be used to identify, isolate, or amplify partialor full-length clones in a deposited library. In some embodiments, thepolynucleotides are genomic or cDNA sequences isolated, or otherwisecomplementary to, a cDNA from a nucleic acid library. Preferably, thecDNA library comprises at least 80% full-length sequences, preferably atleast 85% or 90% full-length sequences, and more preferably at least 95%full-length sequences. The cDNA libraries can be normalized to increasethe representation of rare sequences. Low stringency hybridizationconditions are typically, but not exclusively, employed with sequenceshaving a reduced sequence identity relative to complementary sequences.Moderate and high stringency conditions can optionally be employed forsequences of greater identity. Low stringency conditions allow selectivehybridization of sequences having about 70% sequence identity and can beemployed to identify orthologous or paralogous sequences.

c . Polynucleotides Having at Least 60% Sequence Identity

The present invention further provides isolated nucleic acids comprisingpolynucleotides, wherein the polynucleotides have a specified identityat the nucleotide level to a polynucleotide as disclosed above. In thisregard, the present invention encompasses polynucleotides that have aspecified identity to the polynucleotides discussed above, but are notthe same as the sequence of FIG. 2. The percentage of identity to areference sequence is at least 60% and, rounded upwards to the nearestinteger, can be expressed as an integer selected from the group ofintegers consisting of from 60 to 99. Thus, for example, the percentageof identity to a reference sequence can be at least 70%, 75%, 80%, 85%,90%, or 95%.

4. Vectors

According to the present invention, the polynucleotide sequence encodingthe CaNIK1 protein may be inserted into any suitable yeast vector withany method known to a person who has skill in the art. The vector willtypically be comprised of a polynucleotide encoding the CaNIK1 proteinoperably linked to any suitable promoter which will direct thetranscription of the polynucleotide in the intended host cell. Examplesof suitable promoters include EF1α2 which is a constitutive promoter andis characterized in Sundstrom et al., General Bacteriology, 172:2036-2045 (1990), and PCK1 which is an inducible promoter and ischaracterized in Leuker et al., Gene 192: 235-240 (1997). According tothe present invention, the promoter is operably linked to thepolynucleotide encoding for the CaNIK1 protein and inserted into a yeasttransformation vector.

Yeast vectors are grouped into five general classes according to theirmode of replication in the yeast: YIp, YRp, YCp, YEp, YLp. Comprehensivelaboratory techniques regarding insertion of polynucleotides into yeastvectors can be found in Chapter 13 of Ausubel et al.

5. Bioassay

Another aspect of the invention is a bioassay useful for screeningpharmaceutical candidates which can inhibit pathogenicity in Candidaalbicans. The bioassay is based on assessing a candidate's ability toinhibit expression or functionality of the CaNik1 gene or its geneproduct, which as explained above, is linked to the virulentcharacteristics of the yeast. A bioassay according to the presentinvention comprises the following steps: transformation of cells fromyeast species that exhibit phenotypic switching with a polynucleotideencoding CaNIK1 protein, and a promoter linked to the polynucleotidesegment which can drive protein expression; effecting contact betweenthe yeast cells and a pharmaceutical candidate; and analyzing the effectof the pharmaceutical candidate on inhibition of the expression of theCaNik1 gene. In one embodiment, C. albicans cells harboring a CaNik1deletion are transformed with a suitable construct containing aCaNIK1-encoding polynucleotide, and an operably linked promoter.

A. Transformation of Yeast Cells

The present invention contemplates the use of yeast cells with aphenotypic switching pathway similar to that of Candida albicans.Srikantha et al., J. Bacteriol. 179: 3837-3844 (1997). Transformation ofthe cells can be accomplished through any means known to a person withskill in the art. One example of a yeast transformation procedure is thelithium acetate procedure whereby yeast cells are briefly incubated inbuffered lithium acetate and transforming DNA is introduced with carrierDNA. Addition of polyethylene glycol (PEG) and a heat shock trigger DNAuptake. An alternate method of transforming yeast cells is theelectroporation procedure whereby concentrated cells are transformedusing an exponential electric pulse. Comprehensive laboratory techniquesregarding yeast transformation procedures can be found in Chapter 13 ofAusubel et al.

B. Contact of a Test Substance with Transformed Cells

According to the present invention, a test substance should make contactwith at least some of a plurality of cells transformed with apolypeptide encoding CaNik1. Contact includes any exposure of the testsubstance to any surface of a transformed cell. A preferred method ofcontact would be incubation of the cells with the test substance.

The test substance includes any compound which may have characteristicsinhibitory to the growth or the pathogenicity of Candida albicans. Anexample of a test substance is a pharmaceutical compound withantimycotic properties.

6. Assessing of the Effect of the Test Substance on CaNik1 GeneExpression

According to the present invention, the effect of the pharmaceuticalcompound on CaNik1 expression is analyzed after contact between thepharmaceutical compound and the plurality of transformed cells. CaNik1expression can be measured through any means known by a person withskill in the art. Examples of methods which monitor the level of geneexpression are: measuring levels of CaNIK1 protein and mRNA produced bythe cells; or measuring the kinase activity within the cell; ormonitoring the level of transcription of a reporter gene operably linkedto a promoter.

An example of monitoring CaNik1 expression is the measurement of levelsof CaNIK1 protein produced by the plurality of cells. This can bemeasured by performing two-dimensional gel electrophoresis using thetechniques of isoelectric-focusing and SDS-polyacrylamide gelelectrophoresis followed by autoradiography of the gel. Comprehensivelaboratory techniques regarding two-dimensional gel electrophoresis andautoradiography can be found in Chapter 10 and Appendix 3 of Ausubel etal.

Another example of monitoring CaNik1 expression is to measure the levelof mRNA encoded within the cell and produced by the plurality. mRNAlevels within the cell can be measured with the following threetechniques: Northern Blot, primer extension and ribonuclease protection.The Northern Blot procedure consists of fractioning mRNA with gelelectrophoresis, transferring the mRNA fragments from the gel onto afilter and hybridizing the target mRNA molecules used a labeled DNA orRNA probe. The primer extension procedure includes hybridizing anoligonucleotide primer to the 5′ end of the target mRNA and extendingthe primer using reverse transcriptase and unlabeled deoxynucleotides toform a single-stranded DNA complementary to the template RNA. Theresultant DNA is analyzed on the sequencing gel. The yield of the primerextension product quantifies the amount of mRNA produced by the cell.The ribonuclease protection assay measures mRNA levels by hybridizingsequence specific RNA probes to sample RNAs. The probe anneals tohomologous sequences in the sample RNA. The presence of target RNA isanalyzed and quantified by gel electrophoresis. Comprehensive laboratorytechniques regarding Northern Blot, primer extension and ribonucleaseprotection assays can be found in Chapter 4 of Ausubel et al.

A third example of monitoring CaNik1 expression is to monitor the levelof kinase activity within the plurality of cells. Kinase activity withinthe cells can be monitored by labeling ATP with ³²p in vitro. Thelabeled ATP acts as the donor substrate, and the CaNIK1 protein acts asthe acceptor substrate. Phosphotransfer is detected as the accumulationof ³²P-labeled protein within the cell. The accumulation of protein ismeasured with polyacrylamide gel electrophoresis and autoradiography.Target kinase activity can be distinguished from background kinaseactivity with autophosphorylation of the CaNIK1 protein onpolyacrylamide gel. Comprehensive laboratory techniques regardingphosphorylation and measurement of kinase activity can be found inChapter 18 of Ausubel et al.

In a further example, a reporter gene is operably linked to a promoterand the level of transcription of the reporter gene is monitored aftercontact between the plurality and the test substance. In accordance withthe present invention, the promoter region of the CaNik1 gene isoperably linked to the luciferase gene. Gene activity is thus linked toluciferase activity, which can then be measured quantitively, with aluminometer, as a bioluminescent reaction.

The present invention is described further below by reference to thefollowing examples, which are illustrative only.

EXAMPLE 1

PCR Amplification to Determine a CaNik1 Probe

The following, deoxyinosine-containing, degenerate primers were designedthat encompassed the highly conserved regions of the two componentresponse regulators LemA (Hrabak & Willis, J Bacteriol 174: 3011-3020(1992)), BarA (Nagasawa et al., Escherichia coli. Mol Microbiol, 6:799-807 (1992)) and SLN1 (Ota & Varshavasky, Science 263: 566-569(1993)), respectively: 1) Slb1: (SEQ ID NO:5)5-GAATTGAGAACGCCTITIAATGG-3, which corresponds to thehistidine-autokinase domain; 2) Slb2: (SEQ ID NO:6) 5-AGTCCTAAGCCAGTACCACC-3, which corresponds to the ATP-binding domain; and 3) Slb3:(SEQ ID NO:7) 5-TTTAGGCATCTGGACITCCAT, which corresponds to the responseregulator domain. Slb1 served as a 5′-end primer for PCR amplifications.The Slb1/Slb2 and Slb1/Slb3 pairs were used to amplify PCR productsusing the Hot-start wax gem (Perkin, Elmer) protocol. The Hot-start waxgem protocol which generates PCR products used the following reactionmixture: 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 1.2 mM MgCl₂, 100 μM dNTP,50 μm of each primer and 2.5 units of Taq polymerase, in a final volumeof 100 μL. Conditions for PCR cycling included denaturation at 94° C.for 1 min, annealing at 40° C. for 1.5 min and extension at 72° C. for2.5 min. For all amplifications, S. cerevisiae genomic DNA was used as acontrol for the amplification of the two component hybrid kinase geneSLN1, to monitor the quality of the PCR products. PCR products were gelpurified and cloned into either PCR-Trap (Hunter Gen) or pGEM T-Easy(Promega Corp.). Three positive clones were chosen for each of the PCRproducts of the two sets of primer pairs. pCN.5/3, pCN.5/11 and pCN.5/21were chosen from the products of Slb1/Slb2; and pCN1.3/5, pCN1.3/13 andpCN 1.3/16 were chosen from the products of Slb1/Slb3.

EXAMPLE 2

Isolation of CaNik1 Gene

To isolate a full-length gene, approximately 8×10⁴plaques of a C.albicans genomic library were screened using a 1.2 kb DNA fragmentisolated from pCN1.3/13, which spanned the histidine-autokinase (H1) andaspartyl receiver domain (D1). Lambda DNA from 20 positive clones wasextracted and Southern blots probed with pCN1.3/13. Using combinationsof primer pairs for the arms of the lambda DNA and either the degenerateprimers for the histidine-autokinase domain (Slb1) or the responseregulator domain (Slb3), lambda clones containing inserts larger than 4kb were identified. The screen was performed with a high fidelity longPCR protocol (Boehringer Mannheim, Inc., Indianapolis, Ind.). Threelambda clones contained DNA fragments larger than 3 kb that flanked theupstream region of the histidine-autokinase domain and the downstreamregion of the aspartyl receiver domain. One of these clones, SA15.1, waschosen to determine the complete nucleotide sequence of the gene in bothdirections using the ABI automated sequencing system and fluorescentdideoxynucleotides as described earlier.

The DNA fragment generated by Slb1/Slb3 was used as a probe to screen aC. albicans EMBL3a lambda genomic library to identify clones containingthe full-length gene. Of 10⁵ pfu's, twenty positive clones wereidentified. Clone λSA15.1, which contained a genomic fragment ofapproximately 4.8 kb with DNA flanking both the H1 and the D domains,was chosen for further characterization. The nucleotide sequence of theDNA insert was determined in both directions. The deduced amino acidsequence revealed an uninterrupted open reading frame of 1081 aminoacids beginning with ATG as the initiation codon. The initiation codonwas surrounded by an atypical Kozak consensus sequence CTCCAATGA, withcytosine at the −3 position (Kozak, Nucleic Acids Res, 12: 857-871(1984)). When total genomic DNA of C. albicans strain WO-1 was digestedwith a variety of restriction enzymes, and the resulting Southern blothybridized under conditions of high stringency (65° C. in Church-Gilberthybridization buffer) (Church & Gilbert, Proc Natl Acad. Sci USA 81:1991-1995 (1984)) with the 1.2 kb probe spanning the 800 bp upstream ofthe gene, the banding pattern suggested that CaNIK1 is encoded by asingle copy gene. When total genomic DNA of strain WO-1 and strain 3153Awas digested with BsaAI or NciI and hybridized with the 4.2 kb probe,the patterns were identical, but when TspI-digested DNA of the twostrains were probed, the patterns differed, suggesting allelicdifferences exist between these strains. A comparison of the CaNik1sequence published recently by Nagahashi et al., Candida albicans.Microbology, 144: 425-432 (1998) for strain IFO1060 and the sequence weobtained for strain WO-1 in the present invention differ at sevennucleotide positions in the open reading frame of 3243 bp.

EXAMPLE 3

Deletion of CaNik1 in C. albicans Strain CAI8

In order to generate a CaNik1 deletion cassette, a DNA fragment ofapproximately 2.1 kb containing both the histidine-autokinase andaspartyl response regulator domains was amplified by PCR using as thetemplate λSA15.1 (FIG. 4a), which contained the 545 bp sequence upstreamof the histidine-autokinase domain. The PCR fragment was gel-purifiedand cloned into the PGEM-T easy vector (Promega). The DNA insert wasagain excised from the recombinant plasmid with EcoRI and subcloned intoa PUC18 vector (Life Technologies) at the EcoRI site. The resultantrecombinant plasmid was designated pUNIK12.1 (FIG. 4b). A deletionconstruct pCNH35 was generated that spanned the histidine-autokinase andATP binding-domains. To construct pCNH35, pUNIK12.1 plasmid DNA (FIG.4b) was digested with AflII and XhoI, and blunt-end repaired with theKlenow DNA polymerase I. The resultant plasmid DNA fragment was then gelpurified and dephosphorylated with shrimp alkaline phosphatase (USBiochemical). A hisG-URA3-hisG cassette of 3.8 kb from pMB9 was thenligated to derive the disruption cassette (FIG. 4c). To isolate theCaNik1 disruption cassette from pCNH35, plasmid DNA was digested withPstI and the digested DNA extracted with phenol: chloroform.Approximately 25 μg of the digestion mixture was used to transformstrain CAI8, an ade2⁻ ura3⁻ derivative of wild type strain SC5314, bythe lithium acetate protocol. Heterozygotes were selected for growth inminimal medium in the absence of uridine. Transformants were initiallytested for the heterozygosity of one of the two CaNik1 alleles bySouthern blot hybridization of genomic DNA digested with PstI. Positiveheterozygotes were further confirmed by digesting genomic DNA with XhoIand by performing Southern blot hybridization. Because the genomicSoutherns revealed polymorphism between the two CaNik1 alleles, twodistinct heterozygotes, NNL6 (L stands for large allele) and NNS7 (Sstands for small allele) were selected. The heterozygote NNS7 was chosento generate the knock-out for the second copy of the CaNIK1 gene. Priorto the knock-out of the second copy, NNS7 was subjected to the 5-FOAselection protocol to convert it from uridine prototrophy to auxotrophy.Loss of the URA3 gene was again confirmed by digestion with XhoI andSouthern blot analysis. In the final step, a single clone, NNS7.1.1,which was heterozygous for the L allele of the CaNik1 locus and URA3⁺,was subjected to a second round of transformation with pCNH35, andselected for growth on defined minimal medium lacking uridine.Transformants which had lost the second copy of CaNik1 were selected bySouthern blot hybridization. One of the 125 transformants obtained withthe pCNH35-based cassette, HH80, contained a homozygous deletion.

EXAMPLE 4

CaNik1 Transcription

To test whether transcription of CaNik1 was regulated by high frequencyphenotypic switching, Northern blots of polyA⁺mRNA of white and opaquephase cell growth cultures of strain WO-1 were probed with the DNAfragment spanning the H1 and ATP binding domains of CaNik1. The CaNik1transcript was detectable at very low levels in both white phase andopaque phase cells throughout the exponential phase of growth and instationary phase. The level of CaNik1 transcript per cell remainedconstant throughout white phase cell growth, but increased steadilyduring opaque phase cell growth, reaching a level per cell roughly twicethat of white phase cells at stationary phase (FIG. 5). Hypha-formingcells of both C. albicans strain WO-1 and C. albicans strain 3153Acontained slightly higher levels of polyA⁺ CaNik1 transcript thanbudding cells. The hypha-to-bud ratio of polyA⁺-containing CaNik1transcript in strain WO-1 and strain 3153A was 1.2 and 1.3,respectively.

EXAMPLE 5

Functional Characterization of the CaNik1 Null Mutant of Strain CAI8

To test whether the CaNik1 deletion mutant HH80 underwent switching, wefirst had to characterize switching in this strain using a low doseultraviolet irradiation protocol that increases switching frequencies.Cells were treated with ultraviolet irradiation for 0, 5, 10, 20 and 40sec, and the percent kill as well as the frequency and type of switchvariants were assessed on modified Lee's medium. The proportions of CAI8and HH80 cells killed after 5, 10, 20, and 40 sec were similar.Identical variant phenotypes were stimulated by UV in both CAI8 and thehomozygous deletion strain HH80. However, the frequency of variantsinduced by comparable levels of UV-irradiation was consistently lower instrain HH80, and this was true in a repeat experiment. For instance, 20sec of UV irradiation resulted in 10.6% and 2.6% variants in CAI8 andHH80 cells, respectively. These results demonstrate that the CaNik1 geneproduct modulates phenotypic switching.

Since deletion of the nik-1⁺ gene in N. crassa affects the morphology ofhyphae, especially at high osmotic strength (Alex et al., Proc Natl AcadSci USA, 93: 3416-3421 (1996), the capability of the CaNik1-minus HH80strain to form hyphae and the morphology of those hyphae were comparedto that of the parent strain CAI8 and a URA3⁺ isogenic strain CAI8U5 at0, 1.0 and 1.5 M NaCl. Under the regime of pH-regulated dimorphism,CAI8, CAI8U5, and HH80 cells formed buds at pH 4.5 and hyphae at pH 6.7.The kinetics of evagination for the three strains at low and high pHwere similar at the three tested salt concentrations. At 1.5 M NaCl, theproportion of cells that formed evaginations at low and high pH wasdramatically reduced in all three strains. The morphology of the hyphaethat formed at pH 6.7 at 0, 1.0, and 1.5 M NaCl were comparable in thethree strains. However, there was a significant and reproducible lag inhyphal growth at 1.5M NaCl in HH80 after 300 min. These resultsdemonstrate that the CaNik1 gene product is not essential for hyphaformation under the regime of pH regulated dimorphism, but its presenceenhances hypha formation at high ionic strength.

Finally, growth of the CaNik1 deletion mutant HH80 was tested at 25° C.and 37° C. for differential sensitivity to osmotic strength and avariety of inhibitors. Patches of budding cells of CAI8, CAI8U5 and HH80were plated on agar containing modified Lee's medium alone or with oneof the following ingredients: 1.0 or 1.5M NaCl; 1M sorbitol; 0.8M KCl;0.5M Mg₂SO₄; 20 or 40 μg per ml calcofluor; 1, 2 or 4 mg per mlcaffeine; 10 or 20 mg per ml hygromycin; 0.002 or 0.004 μg per mlechinocandin; and 0.2 or 0.4M polymyxin B. In three independentexperiments, no qualitative differences were observed between the growthof the control strains and the mutant strain HH80 for any of the testedconditions.

All publications and patent applications referred to in thisspecification are indicative of the level of skill of those in the artto which the invention pertains.

Other objects, features and advantages of the present invention willbecome apparent from the foregoing detailed description and examples. Itshould be understood, however, that the detailed description and thespecific examples, while indicating preferred embodiments of theinvention, are given only by way of illustration.

TABLE 1 Conditions used to test the effect of gene deletion *Phenotypiceffect in HH80 1. Growth kinetics in a) Lee's modified broth Similar toSC5314, CAI8U5, and CAI8 b) YPD broth Similar to SC5314, CAI8U5, andCAI8²⁰. 2. Growth on agar plates with Lee's modified medium or YPD brothsupplemented with: a) None ++++ b) 1 M NaCl ++ c) 1.5 M NaCl + d) 1 MKCl ++ e) 1.2 M Sorbitol ++++ f) 0.5 m MgSO₄ ++ g) Caffeine (1-4 mg/mL)v h) Calcofluor (20-40 μg/mL) +++^(v) i) Echinocandin (0.002-0.004μg/mL) ± j) 2% Trehalose ++++* k) 2% Raffinose ++++* l) 1 M Xylitol ++++m) 10% Glycerol ++++* 3. Switching a) spontaneous frequency No effect b)UV-stimulated frequency Decreased c) repertoire of switch phenoype Noeffect 4. Hypha-induction under the regime of pH-regulated dimorphism.with no osmotic shock: a) time for 50% evagination No effect b)morphology of hypha No effect c) growth of hyphal filaments with osmoticshock using 1.5 M NaCl   i) time for 50% evaginations decreased in bothwild type and the mutant   ii) morphology of hyphac no differencebetween wild hyphae and the mutant   iii) growth of hyphal filaments thegrowth of the hyphae after 300 min was reduced in the mutant as comparedto that in wild type In order to asses the effect of gene deletion ongrowth, exponentially grown cells of wild type (SC53 14), parentalauxotrophic strain used to delete NIK1 gene (CAI8), URA3⁺ derivative ofCAI8 (CAI8U5) and homozygous deletion mutant (HH80) were seriallydiluted and spot plated on agar plates with or without supplements inthe medium. In all the growth medium used in this study, 2% glucoseserved as a carbon source except in the # growth medium containingraffinose, trehalose and glycerol. The symbol “v” denote variablegrowth. Growth of the cultures were qualitatively assessed as very good(++++), good (+++), fair (++), poor (+), poor to no growth (±). 0indicates that colonies were very small (less than 1mm) as assessed bythe colony size on agar plates spread with cultures to generate 50 to100 individual colonies. The growth of the cultures were # assessedafter 2 or 3 days incubation both at 25° C. and 37° C.

7 1 1254 DNA Candida albicans CDS (1)..(1254) 1 gag att aga aca cca ttgaat ggg att att ggw atg acy cag ttg tcr 48 Glu Ile Arg Thr Pro Leu AsnGly Ile Ile Gly Met Thr Gln Leu Ser 1 5 10 15 ctt gat aca gag ttg acrcag tac caa cga gag atg ttg tcg att gtg 96 Leu Asp Thr Glu Leu Thr GlnTyr Gln Arg Glu Met Leu Ser Ile Val 20 25 30 cat aac ttg gca aat tcc ttgttg acc att ata gac gat ata ttg gat 144 His Asn Leu Ala Asn Ser Leu LeuThr Ile Ile Asp Asp Ile Leu Asp 35 40 45 att tct aag att gag gcg aat agaatg acg gtg gaa cag att gat ttt 192 Ile Ser Lys Ile Glu Ala Asn Arg MetThr Val Glu Gln Ile Asp Phe 50 55 60 tca tta aga ggg aca gtg ttt ggt gcattg aaa acg tta gcc gtc aaa 240 Ser Leu Arg Gly Thr Val Phe Gly Ala LeuLys Thr Leu Ala Val Lys 65 70 75 80 gct att gaa aaa aac cta gac ttg acctat caa tgt gat tca tcg ttt 288 Ala Ile Glu Lys Asn Leu Asp Leu Thr TyrGln Cys Asp Ser Ser Phe 85 90 95 cca gat aat ctt att gga gat agt ttt agatta cga caa gtt att ctt 336 Pro Asp Asn Leu Ile Gly Asp Ser Phe Arg LeuArg Gln Val Ile Leu 100 105 110 aac ttg gct ggt aat gct att aag ttt actaaa gag ggg aaa gtt agt 384 Asn Leu Ala Gly Asn Ala Ile Lys Phe Thr LysGlu Gly Lys Val Ser 115 120 125 gtt agt gtg aaa aag tct gat aaa atg gtgtta gat agt aag ttg ttg 432 Val Ser Val Lys Lys Ser Asp Lys Met Val LeuAsp Ser Lys Leu Leu 130 135 140 tta gag gtt tgt gtt agc gac acg gga ataggt ata gag aaa gac aaa 480 Leu Glu Val Cys Val Ser Asp Thr Gly Ile GlyIle Glu Lys Asp Lys 145 150 155 160 ttg gga ttg att ttc gat acc ttc tgtcaa gct gat ggt tct act aca 528 Leu Gly Leu Ile Phe Asp Thr Phe Cys GlnAla Asp Gly Ser Thr Thr 165 170 175 aga aag ttt ggt ggt aca ggt tta gggttg tca att tcc aaa cag ttg 576 Arg Lys Phe Gly Gly Thr Gly Leu Gly LeuSer Ile Ser Lys Gln Leu 180 185 190 ata cat tta atg ggt gga gag ata tgggtt act tcg gag tat gga tcc 624 Ile His Leu Met Gly Gly Glu Ile Trp ValThr Ser Glu Tyr Gly Ser 195 200 205 ggr tca aac ttt tat ttt acg gtg tgcgtg tcg cca tct aat att aga 672 Gly Ser Asn Phe Tyr Phe Thr Val Cys ValSer Pro Ser Asn Ile Arg 210 215 220 tat act cga caa acc gaa caa ttg ttacca ttt agt tcc cat tat gtg 720 Tyr Thr Arg Gln Thr Glu Gln Leu Leu ProPhe Ser Ser His Tyr Val 225 230 235 240 tta ttt gta tcg act gag cat actcaa gaa gaa ctt gat gtg ttg aga 768 Leu Phe Val Ser Thr Glu His Thr GlnGlu Glu Leu Asp Val Leu Arg 245 250 255 gat gga att ata gaa ctt gga ttgata cct ata ata gtg aga aat att 816 Asp Gly Ile Ile Glu Leu Gly Leu IlePro Ile Ile Val Arg Asn Ile 260 265 270 gaa gat gca aca ttg act gag ccggtg aaa tat gat ata att atg att 864 Glu Asp Ala Thr Leu Thr Glu Pro ValLys Tyr Asp Ile Ile Met Ile 275 280 285 gat tcg ata gag att gcc aaa aagttg agg ttg tta tcg gag gtt aaa 912 Asp Ser Ile Glu Ile Ala Lys Lys LeuArg Leu Leu Ser Glu Val Lys 290 295 300 tat att ccg ttg gtt ttg gtc catcat tct att cca cag ttg aat atg 960 Tyr Ile Pro Leu Val Leu Val His HisSer Ile Pro Gln Leu Asn Met 305 310 315 320 aga gta tgt att gat ttg gggata tct tcc tat gca aat acg cca tgt 1008 Arg Val Cys Ile Asp Leu Gly IleSer Ser Tyr Ala Asn Thr Pro Cys 325 330 335 tcg atc acg gac ttg gcc agtgcg att ata cca gcg ttg gag tcg aga 1056 Ser Ile Thr Asp Leu Ala Ser AlaIle Ile Pro Ala Leu Glu Ser Arg 340 345 350 tct ata tca cag aac tca gacgag tcg gtg agg tac aaa ata tta cta 1104 Ser Ile Ser Gln Asn Ser Asp GluSer Val Arg Tyr Lys Ile Leu Leu 355 360 365 gca gag gac aac ctc gtc aatcag aaa ctt gca gtt agg ata tta gaa 1152 Ala Glu Asp Asn Leu Val Asn GlnLys Leu Ala Val Arg Ile Leu Glu 370 375 380 aag caa ggg cat ctg gtg gaagta gtt gag aac gga ctc gag gcg tac 1200 Lys Gln Gly His Leu Val Glu ValVal Glu Asn Gly Leu Glu Ala Tyr 385 390 395 400 gaa gcg att aag agg aataaa tat gat gtg gtg ttg atg gat gtg caa 1248 Glu Ala Ile Lys Arg Asn LysTyr Asp Val Val Leu Met Asp Val Gln 405 410 415 atg cct 1254 Met Pro 2418 PRT Candida albicans 2 Glu Ile Arg Thr Pro Leu Asn Gly Ile Ile GlyMet Thr Gln Leu Ser 1 5 10 15 Leu Asp Thr Glu Leu Thr Gln Tyr Gln ArgGlu Met Leu Ser Ile Val 20 25 30 His Asn Leu Ala Asn Ser Leu Leu Thr IleIle Asp Asp Ile Leu Asp 35 40 45 Ile Ser Lys Ile Glu Ala Asn Arg Met ThrVal Glu Gln Ile Asp Phe 50 55 60 Ser Leu Arg Gly Thr Val Phe Gly Ala LeuLys Thr Leu Ala Val Lys 65 70 75 80 Ala Ile Glu Lys Asn Leu Asp Leu ThrTyr Gln Cys Asp Ser Ser Phe 85 90 95 Pro Asp Asn Leu Ile Gly Asp Ser PheArg Leu Arg Gln Val Ile Leu 100 105 110 Asn Leu Ala Gly Asn Ala Ile LysPhe Thr Lys Glu Gly Lys Val Ser 115 120 125 Val Ser Val Lys Lys Ser AspLys Met Val Leu Asp Ser Lys Leu Leu 130 135 140 Leu Glu Val Cys Val SerAsp Thr Gly Ile Gly Ile Glu Lys Asp Lys 145 150 155 160 Leu Gly Leu IlePhe Asp Thr Phe Cys Gln Ala Asp Gly Ser Thr Thr 165 170 175 Arg Lys PheGly Gly Thr Gly Leu Gly Leu Ser Ile Ser Lys Gln Leu 180 185 190 Ile HisLeu Met Gly Gly Glu Ile Trp Val Thr Ser Glu Tyr Gly Ser 195 200 205 GlySer Asn Phe Tyr Phe Thr Val Cys Val Ser Pro Ser Asn Ile Arg 210 215 220Tyr Thr Arg Gln Thr Glu Gln Leu Leu Pro Phe Ser Ser His Tyr Val 225 230235 240 Leu Phe Val Ser Thr Glu His Thr Gln Glu Glu Leu Asp Val Leu Arg245 250 255 Asp Gly Ile Ile Glu Leu Gly Leu Ile Pro Ile Ile Val Arg AsnIle 260 265 270 Glu Asp Ala Thr Leu Thr Glu Pro Val Lys Tyr Asp Ile IleMet Ile 275 280 285 Asp Ser Ile Glu Ile Ala Lys Lys Leu Arg Leu Leu SerGlu Val Lys 290 295 300 Tyr Ile Pro Leu Val Leu Val His His Ser Ile ProGln Leu Asn Met 305 310 315 320 Arg Val Cys Ile Asp Leu Gly Ile Ser SerTyr Ala Asn Thr Pro Cys 325 330 335 Ser Ile Thr Asp Leu Ala Ser Ala IleIle Pro Ala Leu Glu Ser Arg 340 345 350 Ser Ile Ser Gln Asn Ser Asp GluSer Val Arg Tyr Lys Ile Leu Leu 355 360 365 Ala Glu Asp Asn Leu Val AsnGln Lys Leu Ala Val Arg Ile Leu Glu 370 375 380 Lys Gln Gly His Leu ValGlu Val Val Glu Asn Gly Leu Glu Ala Tyr 385 390 395 400 Glu Ala Ile LysArg Asn Lys Tyr Asp Val Val Leu Met Asp Val Gln 405 410 415 Met Pro 33246 DNA Candida albicans CDS (1)..(3243) 3 atg aac ccc act aaa aaa cctcgg tta tca cca atg cag ccc tct gtt 48 Met Asn Pro Thr Lys Lys Pro ArgLeu Ser Pro Met Gln Pro Ser Val 1 5 10 15 ttt gaa ata ctc aac gac cctgag ctt tat agt cag cac tgt cat agc 96 Phe Glu Ile Leu Asn Asp Pro GluLeu Tyr Ser Gln His Cys His Ser 20 25 30 ctt agg gaa aca ctt ctt gat catttc aac cat caa gct aca ctt atc 144 Leu Arg Glu Thr Leu Leu Asp His PheAsn His Gln Ala Thr Leu Ile 35 40 45 gac act tat gaa cat gaa cta gaa aaatcc aaa aat gcc aac aaa gcg 192 Asp Thr Tyr Glu His Glu Leu Glu Lys SerLys Asn Ala Asn Lys Ala 50 55 60 tcc caa caa gca ctt agt gaa ata ggt acagtt gtt ata tct gtt gcc 240 Ser Gln Gln Ala Leu Ser Glu Ile Gly Thr ValVal Ile Ser Val Ala 65 70 75 80 atg gga gac ttg tcg aaa aaa gtt gag attcac aca gta gaa aat gac 288 Met Gly Asp Leu Ser Lys Lys Val Glu Ile HisThr Val Glu Asn Asp 85 90 95 cct gag att tta aaa gtc aaa atc acc atc aacacc atg atg gat caa 336 Pro Glu Ile Leu Lys Val Lys Ile Thr Ile Asn ThrMet Met Asp Gln 100 105 110 tta cag aca ttt gct aat gag gtt aca aaa gtcgcc acc gaa gtc gca 384 Leu Gln Thr Phe Ala Asn Glu Val Thr Lys Val AlaThr Glu Val Ala 115 120 125 aat ggt gaa cta ggt gga caa gcg aaa aat gatgga tct gtt ggt att 432 Asn Gly Glu Leu Gly Gly Gln Ala Lys Asn Asp GlySer Val Gly Ile 130 135 140 tgg aga tca ctt aca gac aat gtt aat att atggct ctt aat tta act 480 Trp Arg Ser Leu Thr Asp Asn Val Asn Ile Met AlaLeu Asn Leu Thr 145 150 155 160 aac caa gtg cga gaa att gct gat gtc acacgt gct gtt gcc aag ggg 528 Asn Gln Val Arg Glu Ile Ala Asp Val Thr ArgAla Val Ala Lys Gly 165 170 175 gac ttg tca cgt aaa att aat gta cac gcccag ggt gaa atc ctt caa 576 Asp Leu Ser Arg Lys Ile Asn Val His Ala GlnGly Glu Ile Leu Gln 180 185 190 ctt caa cgt aca ata aac acc atg gtg gatcag tta cga acg ttt gca 624 Leu Gln Arg Thr Ile Asn Thr Met Val Asp GlnLeu Arg Thr Phe Ala 195 200 205 ttc gaa gta tct aaa gtt gct aga gat gttggt gtg ctt ggt ata tta 672 Phe Glu Val Ser Lys Val Ala Arg Asp Val GlyVal Leu Gly Ile Leu 210 215 220 gga gga caa gcg ttg att gaa aat gtt gaaggt att tgg gaa gag ttg 720 Gly Gly Gln Ala Leu Ile Glu Asn Val Glu GlyIle Trp Glu Glu Leu 225 230 235 240 act gat aat gtc aat gcc atg gct cttaat ttg act aca caa gtg aga 768 Thr Asp Asn Val Asn Ala Met Ala Leu AsnLeu Thr Thr Gln Val Arg 245 250 255 aat att gcc aat gtc acc act gcc gttgcc aag ggg gat ttg tcg aaa 816 Asn Ile Ala Asn Val Thr Thr Ala Val AlaLys Gly Asp Leu Ser Lys 260 265 270 aaa gtc act gct gat tgt aag gga gaaaty ctt gat ttg aaa ctt act 864 Lys Val Thr Ala Asp Cys Lys Gly Glu IleLeu Asp Leu Lys Leu Thr 275 280 285 att aat caa atg gtg gac cga tta cagaat ttt gct ctt gcg gtg acg 912 Ile Asn Gln Met Val Asp Arg Leu Gln AsnPhe Ala Leu Ala Val Thr 290 295 300 aca ttg tcg aga gag gtt ggt act ttgggt att ttg ggt gga caa gct 960 Thr Leu Ser Arg Glu Val Gly Thr Leu GlyIle Leu Gly Gly Gln Ala 305 310 315 320 aac gta cag gat gtt gaa ggt gcttgg aaa cag gtt aca gaa aat gtc 1008 Asn Val Gln Asp Val Glu Gly Ala TrpLys Gln Val Thr Glu Asn Val 325 330 335 aac cta atg gct act aat tta actaac caa gtg aga tct att gct aca 1056 Asn Leu Met Ala Thr Asn Leu Thr AsnGln Val Arg Ser Ile Ala Thr 340 345 350 gtt act act gca gtt gcg cat ggtgat ttg tcg caa aag att gat ggt 1104 Val Thr Thr Ala Val Ala His Gly AspLeu Ser Gln Lys Ile Asp Gly 355 360 365 cat ccc aaa gga gag att tta caattg aaa aat aca atc aac aag atg 1152 His Pro Lys Gly Glu Ile Leu Gln LeuLys Asn Thr Ile Asn Lys Met 370 375 380 gtg gac tct ttg cag ttg ttt gcatca gaa gtg tcg aaa gtg gca caa 1200 Val Asp Ser Leu Gln Leu Phe Ala SerGlu Val Ser Lys Val Ala Gln 385 390 395 400 gat gtt ggt att aat gga aaatta ggt att caa gca caa gtt agt gat 1248 Asp Val Gly Ile Asn Gly Lys LeuGly Ile Gln Ala Gln Val Ser Asp 405 410 415 gtt gat gga tta tgg aag gagatt acg tct aat gta aat acc atg gct 1296 Val Asp Gly Leu Trp Lys Glu IleThr Ser Asn Val Asn Thr Met Ala 420 425 430 tca aat tta act tcg caa gtgaga gct ttt gca cag att act gct gct 1344 Ser Asn Leu Thr Ser Gln Val ArgAla Phe Ala Gln Ile Thr Ala Ala 435 440 445 gct act gat ggg gat ttc actaga ttt att act gtt gaa gca ctg gga 1392 Ala Thr Asp Gly Asp Phe Thr ArgPhe Ile Thr Val Glu Ala Leu Gly 450 455 460 gag atg gat gcg ttg aaa acaaag att aat caa atg gtg ttt aac tta 1440 Glu Met Asp Ala Leu Lys Thr LysIle Asn Gln Met Val Phe Asn Leu 465 470 475 480 agg gaa tcg ctt caa aggaat act gcg gct aga gaa gct gct gag ttg 1488 Arg Glu Ser Leu Gln Arg AsnThr Ala Ala Arg Glu Ala Ala Glu Leu 485 490 495 gcc aat agt gcg aaa tccgag ttt tta gca aac atg tcg cat gag att 1536 Ala Asn Ser Ala Lys Ser GluPhe Leu Ala Asn Met Ser His Glu Ile 500 505 510 aga aca cca ttg aat gggatt att ggw atg acy cag ttg tcr ctt gat 1584 Arg Thr Pro Leu Asn Gly IleIle Gly Met Thr Gln Leu Ser Leu Asp 515 520 525 aca gag ttg acr cag taccaa cga gag atg ttg tcg att gtg cat aac 1632 Thr Glu Leu Thr Gln Tyr GlnArg Glu Met Leu Ser Ile Val His Asn 530 535 540 ttg gca aat tcc ttg ttgacc att ata gac gat ata ttg gat att tct 1680 Leu Ala Asn Ser Leu Leu ThrIle Ile Asp Asp Ile Leu Asp Ile Ser 545 550 555 560 aag att gag gcg aataga atg acg gtg gaa cag att gat ttt tca tta 1728 Lys Ile Glu Ala Asn ArgMet Thr Val Glu Gln Ile Asp Phe Ser Leu 565 570 575 aga ggg aca gtg tttggt gca ttg aaa acg tta gcc gtc aaa gct att 1776 Arg Gly Thr Val Phe GlyAla Leu Lys Thr Leu Ala Val Lys Ala Ile 580 585 590 gaa aaa aac cta gacttg acc tat caa tgt gat tca tcg ttt cca gat 1824 Glu Lys Asn Leu Asp LeuThr Tyr Gln Cys Asp Ser Ser Phe Pro Asp 595 600 605 aat ctt att gga gatagt ttt aga tta cga caa gtt att ctt aac ttg 1872 Asn Leu Ile Gly Asp SerPhe Arg Leu Arg Gln Val Ile Leu Asn Leu 610 615 620 gct ggt aat gct attaag ttt act aaa gag ggg aaa gtt agt gtt agt 1920 Ala Gly Asn Ala Ile LysPhe Thr Lys Glu Gly Lys Val Ser Val Ser 625 630 635 640 gtg aaa aag tctgat aaa atg gtg tta gat agt aag ttg ttg tta gag 1968 Val Lys Lys Ser AspLys Met Val Leu Asp Ser Lys Leu Leu Leu Glu 645 650 655 gtt tgt gtt agcgac acg gga ata ggt ata gag aaa gac aaa ttg gga 2016 Val Cys Val Ser AspThr Gly Ile Gly Ile Glu Lys Asp Lys Leu Gly 660 665 670 ttg att ttc gatacc ttc tgt caa gct gat ggt tct act aca aga aag 2064 Leu Ile Phe Asp ThrPhe Cys Gln Ala Asp Gly Ser Thr Thr Arg Lys 675 680 685 ttt ggt ggt acaggt tta ggg ttg tca att tcc aaa cag ttg ata cat 2112 Phe Gly Gly Thr GlyLeu Gly Leu Ser Ile Ser Lys Gln Leu Ile His 690 695 700 tta atg ggt ggagag ata tgg gtt act tcg gag tat gga tcc ggr tca 2160 Leu Met Gly Gly GluIle Trp Val Thr Ser Glu Tyr Gly Ser Gly Ser 705 710 715 720 aac ttt tatttt acg gtg tgc gtg tcg cca tct aat att aga tat act 2208 Asn Phe Tyr PheThr Val Cys Val Ser Pro Ser Asn Ile Arg Tyr Thr 725 730 735 cga caa accgaa caa ttg tta cca ttt agt tcc cat tat gtg tta ttt 2256 Arg Gln Thr GluGln Leu Leu Pro Phe Ser Ser His Tyr Val Leu Phe 740 745 750 gta tcg actgag cat act caa gaa gaa ctt gat gtg ttg aga gat gga 2304 Val Ser Thr GluHis Thr Gln Glu Glu Leu Asp Val Leu Arg Asp Gly 755 760 765 att ata gaactt gga ttg ata cct ata ata gtg aga aat att gaa gat 2352 Ile Ile Glu LeuGly Leu Ile Pro Ile Ile Val Arg Asn Ile Glu Asp 770 775 780 gca aca ttgact gag ccg gtg aaa tat gat ata att atg att gat tcg 2400 Ala Thr Leu ThrGlu Pro Val Lys Tyr Asp Ile Ile Met Ile Asp Ser 785 790 795 800 ata gagatt gcc aaa aag ttg agg ttg tta tcg gag gtt aaa tat att 2448 Ile Glu IleAla Lys Lys Leu Arg Leu Leu Ser Glu Val Lys Tyr Ile 805 810 815 ccg ttggtt ttg gtc cat cat tct att cca cag ttg aat atg aga gta 2496 Pro Leu ValLeu Val His His Ser Ile Pro Gln Leu Asn Met Arg Val 820 825 830 tgt attgat ttg ggg ata tct tcc tat gca aat acg cca tgt tcg atc 2544 Cys Ile AspLeu Gly Ile Ser Ser Tyr Ala Asn Thr Pro Cys Ser Ile 835 840 845 acg gacttg gcc agt gcg att ata cca gcg ttg gag tcg aga tct ata 2592 Thr Asp LeuAla Ser Ala Ile Ile Pro Ala Leu Glu Ser Arg Ser Ile 850 855 860 tca cagaac tca gac gag tcg gtg agg tac aaa ata tta cta gca gag 2640 Ser Gln AsnSer Asp Glu Ser Val Arg Tyr Lys Ile Leu Leu Ala Glu 865 870 875 880 gacaac ctc gtc aat cag aaa ctt gca gtt agg ata tta gaa aag caa 2688 Asp AsnLeu Val Asn Gln Lys Leu Ala Val Arg Ile Leu Glu Lys Gln 885 890 895 gggcat ctg gtg gaa gta gtt gag aac gga ctc gag gcg tac gaa gcg 2736 Gly HisLeu Val Glu Val Val Glu Asn Gly Leu Glu Ala Tyr Glu Ala 900 905 910 attaag agg aat aaa tat gat gtg gtg ttg atg gat gtg caa atg cct 2784 Ile LysArg Asn Lys Tyr Asp Val Val Leu Met Asp Val Gln Met Pro 915 920 925 gtaatg ggt ggg ttt gaa gct acg gag aag att cga caa tgg gag aaa 2832 Val MetGly Gly Phe Glu Ala Thr Glu Lys Ile Arg Gln Trp Glu Lys 930 935 940 aagtct aac cca att gac tcg ttg acc ttt agg act cca att att gcc 2880 Lys SerAsn Pro Ile Asp Ser Leu Thr Phe Arg Thr Pro Ile Ile Ala 945 950 955 960ctc act gca cac gcc atg tta ggt gat aga gaa aag tca ttg gcc aag 2928 LeuThr Ala His Ala Met Leu Gly Asp Arg Glu Lys Ser Leu Ala Lys 965 970 975ggg atg gac gat tat gtg agt aag cca ttg aag ccg aaa ttg tta atg 2976 GlyMet Asp Asp Tyr Val Ser Lys Pro Leu Lys Pro Lys Leu Leu Met 980 985 990cag acg ata aag aag tgt att cat aat att aac cag ttg aaa gaa ttg 3024 GlnThr Ile Lys Lys Cys Ile His Asn Ile Asn Gln Leu Lys Glu Leu 995 10001005 tcg aga aat agt agg ggt agc gat ttt gca aag aag atg acc cga aac3072 Ser Arg Asn Ser Arg Gly Ser Asp Phe Ala Lys Lys Met Thr Arg Asn1010 1015 1020 aca ccc ggc cgc acg acc cgt cag ggg agt gat gag ggg agtgta aag 3120 Thr Pro Gly Arg Thr Thr Arg Gln Gly Ser Asp Glu Gly Ser ValLys 1025 1030 1035 1040 gac atg att ggg gac act ccc cgt caa ggg agt gtggag gga ggg ggt 3168 Asp Met Ile Gly Asp Thr Pro Arg Gln Gly Ser Val GluGly Gly Gly 1045 1050 1055 aca agt agt aga cca gta cag aga agg tct gccagg gag ggg tcg atc 3216 Thr Ser Ser Arg Pro Val Gln Arg Arg Ser Ala ArgGlu Gly Ser Ile 1060 1065 1070 act aca att agt gaa caa atc gac cgt tag3246 Thr Thr Ile Ser Glu Gln Ile Asp Arg 1075 1080 4 1081 PRT Candidaalbicans 4 Met Asn Pro Thr Lys Lys Pro Arg Leu Ser Pro Met Gln Pro SerVal 1 5 10 15 Phe Glu Ile Leu Asn Asp Pro Glu Leu Tyr Ser Gln His CysHis Ser 20 25 30 Leu Arg Glu Thr Leu Leu Asp His Phe Asn His Gln Ala ThrLeu Ile 35 40 45 Asp Thr Tyr Glu His Glu Leu Glu Lys Ser Lys Asn Ala AsnLys Ala 50 55 60 Ser Gln Gln Ala Leu Ser Glu Ile Gly Thr Val Val Ile SerVal Ala 65 70 75 80 Met Gly Asp Leu Ser Lys Lys Val Glu Ile His Thr ValGlu Asn Asp 85 90 95 Pro Glu Ile Leu Lys Val Lys Ile Thr Ile Asn Thr MetMet Asp Gln 100 105 110 Leu Gln Thr Phe Ala Asn Glu Val Thr Lys Val AlaThr Glu Val Ala 115 120 125 Asn Gly Glu Leu Gly Gly Gln Ala Lys Asn AspGly Ser Val Gly Ile 130 135 140 Trp Arg Ser Leu Thr Asp Asn Val Asn IleMet Ala Leu Asn Leu Thr 145 150 155 160 Asn Gln Val Arg Glu Ile Ala AspVal Thr Arg Ala Val Ala Lys Gly 165 170 175 Asp Leu Ser Arg Lys Ile AsnVal His Ala Gln Gly Glu Ile Leu Gln 180 185 190 Leu Gln Arg Thr Ile AsnThr Met Val Asp Gln Leu Arg Thr Phe Ala 195 200 205 Phe Glu Val Ser LysVal Ala Arg Asp Val Gly Val Leu Gly Ile Leu 210 215 220 Gly Gly Gln AlaLeu Ile Glu Asn Val Glu Gly Ile Trp Glu Glu Leu 225 230 235 240 Thr AspAsn Val Asn Ala Met Ala Leu Asn Leu Thr Thr Gln Val Arg 245 250 255 AsnIle Ala Asn Val Thr Thr Ala Val Ala Lys Gly Asp Leu Ser Lys 260 265 270Lys Val Thr Ala Asp Cys Lys Gly Glu Ile Leu Asp Leu Lys Leu Thr 275 280285 Ile Asn Gln Met Val Asp Arg Leu Gln Asn Phe Ala Leu Ala Val Thr 290295 300 Thr Leu Ser Arg Glu Val Gly Thr Leu Gly Ile Leu Gly Gly Gln Ala305 310 315 320 Asn Val Gln Asp Val Glu Gly Ala Trp Lys Gln Val Thr GluAsn Val 325 330 335 Asn Leu Met Ala Thr Asn Leu Thr Asn Gln Val Arg SerIle Ala Thr 340 345 350 Val Thr Thr Ala Val Ala His Gly Asp Leu Ser GlnLys Ile Asp Gly 355 360 365 His Pro Lys Gly Glu Ile Leu Gln Leu Lys AsnThr Ile Asn Lys Met 370 375 380 Val Asp Ser Leu Gln Leu Phe Ala Ser GluVal Ser Lys Val Ala Gln 385 390 395 400 Asp Val Gly Ile Asn Gly Lys LeuGly Ile Gln Ala Gln Val Ser Asp 405 410 415 Val Asp Gly Leu Trp Lys GluIle Thr Ser Asn Val Asn Thr Met Ala 420 425 430 Ser Asn Leu Thr Ser GlnVal Arg Ala Phe Ala Gln Ile Thr Ala Ala 435 440 445 Ala Thr Asp Gly AspPhe Thr Arg Phe Ile Thr Val Glu Ala Leu Gly 450 455 460 Glu Met Asp AlaLeu Lys Thr Lys Ile Asn Gln Met Val Phe Asn Leu 465 470 475 480 Arg GluSer Leu Gln Arg Asn Thr Ala Ala Arg Glu Ala Ala Glu Leu 485 490 495 AlaAsn Ser Ala Lys Ser Glu Phe Leu Ala Asn Met Ser His Glu Ile 500 505 510Arg Thr Pro Leu Asn Gly Ile Ile Gly Met Thr Gln Leu Ser Leu Asp 515 520525 Thr Glu Leu Thr Gln Tyr Gln Arg Glu Met Leu Ser Ile Val His Asn 530535 540 Leu Ala Asn Ser Leu Leu Thr Ile Ile Asp Asp Ile Leu Asp Ile Ser545 550 555 560 Lys Ile Glu Ala Asn Arg Met Thr Val Glu Gln Ile Asp PheSer Leu 565 570 575 Arg Gly Thr Val Phe Gly Ala Leu Lys Thr Leu Ala ValLys Ala Ile 580 585 590 Glu Lys Asn Leu Asp Leu Thr Tyr Gln Cys Asp SerSer Phe Pro Asp 595 600 605 Asn Leu Ile Gly Asp Ser Phe Arg Leu Arg GlnVal Ile Leu Asn Leu 610 615 620 Ala Gly Asn Ala Ile Lys Phe Thr Lys GluGly Lys Val Ser Val Ser 625 630 635 640 Val Lys Lys Ser Asp Lys Met ValLeu Asp Ser Lys Leu Leu Leu Glu 645 650 655 Val Cys Val Ser Asp Thr GlyIle Gly Ile Glu Lys Asp Lys Leu Gly 660 665 670 Leu Ile Phe Asp Thr PheCys Gln Ala Asp Gly Ser Thr Thr Arg Lys 675 680 685 Phe Gly Gly Thr GlyLeu Gly Leu Ser Ile Ser Lys Gln Leu Ile His 690 695 700 Leu Met Gly GlyGlu Ile Trp Val Thr Ser Glu Tyr Gly Ser Gly Ser 705 710 715 720 Asn PheTyr Phe Thr Val Cys Val Ser Pro Ser Asn Ile Arg Tyr Thr 725 730 735 ArgGln Thr Glu Gln Leu Leu Pro Phe Ser Ser His Tyr Val Leu Phe 740 745 750Val Ser Thr Glu His Thr Gln Glu Glu Leu Asp Val Leu Arg Asp Gly 755 760765 Ile Ile Glu Leu Gly Leu Ile Pro Ile Ile Val Arg Asn Ile Glu Asp 770775 780 Ala Thr Leu Thr Glu Pro Val Lys Tyr Asp Ile Ile Met Ile Asp Ser785 790 795 800 Ile Glu Ile Ala Lys Lys Leu Arg Leu Leu Ser Glu Val LysTyr Ile 805 810 815 Pro Leu Val Leu Val His His Ser Ile Pro Gln Leu AsnMet Arg Val 820 825 830 Cys Ile Asp Leu Gly Ile Ser Ser Tyr Ala Asn ThrPro Cys Ser Ile 835 840 845 Thr Asp Leu Ala Ser Ala Ile Ile Pro Ala LeuGlu Ser Arg Ser Ile 850 855 860 Ser Gln Asn Ser Asp Glu Ser Val Arg TyrLys Ile Leu Leu Ala Glu 865 870 875 880 Asp Asn Leu Val Asn Gln Lys LeuAla Val Arg Ile Leu Glu Lys Gln 885 890 895 Gly His Leu Val Glu Val ValGlu Asn Gly Leu Glu Ala Tyr Glu Ala 900 905 910 Ile Lys Arg Asn Lys TyrAsp Val Val Leu Met Asp Val Gln Met Pro 915 920 925 Val Met Gly Gly PheGlu Ala Thr Glu Lys Ile Arg Gln Trp Glu Lys 930 935 940 Lys Ser Asn ProIle Asp Ser Leu Thr Phe Arg Thr Pro Ile Ile Ala 945 950 955 960 Leu ThrAla His Ala Met Leu Gly Asp Arg Glu Lys Ser Leu Ala Lys 965 970 975 GlyMet Asp Asp Tyr Val Ser Lys Pro Leu Lys Pro Lys Leu Leu Met 980 985 990Gln Thr Ile Lys Lys Cys Ile His Asn Ile Asn Gln Leu Lys Glu Leu 995 10001005 Ser Arg Asn Ser Arg Gly Ser Asp Phe Ala Lys Lys Met Thr Arg Asn1010 1015 1020 Thr Pro Gly Arg Thr Thr Arg Gln Gly Ser Asp Glu Gly SerVal Lys 1025 1030 1035 1040 Asp Met Ile Gly Asp Thr Pro Arg Gln Gly SerVal Glu Gly Gly Gly 1045 1050 1055 Thr Ser Ser Arg Pro Val Gln Arg ArgSer Ala Arg Glu Gly Ser Ile 1060 1065 1070 Thr Thr Ile Ser Glu Gln IleAsp Arg 1075 1080 5 23 DNA Artificial Sequence Description of ArtificialSequence Degenerate primer 5 gaattgagaa cgcctntnaa tgg 23 6 20 DNAArtificial Sequence Description of Artificial Sequence Degenerate primer6 agncctaagc cagtaccacc 20 7 21 DNA Artificial Sequence Description ofArtificial Sequence Degenerate primer 7 tttaggcatc tggacntcca t 21

What is claimed is:
 1. An isolated polynucleotide that encodes a proteinlinked to phenotypic switching in Candida albicans that exhibits 70% orgreater overall sequence identity to SEQ. ID No. 3, wherein said proteindisplays kinase activity.
 2. The polynucleotide of claim 1 that exhibits80% or greater identity to SEQ ID No
 3. 3. The polynucleotide of claim 1that exhibits 90% or greater identity to SEQ ID NO
 3. 4. Apolynucleotide according to claim 1, comprising the sequence of SEQ IDNo.
 3. 5. A method of screening for a compound with the ability toinhibit expression or functionality of the CaNIK1 protein comprising:(A) contacting a yeast cell that exhibits phenotypic switching with atest substance, wherein said yeast cell comprises: (i) a polynucleotideaccording to claim 1 and (ii) a promoter operably linked to saidpolynucleotide, such that said yeast cell produces a protein encoded bysaid polynucleotide; then (B) monitoring the ability of said testsubstance to inhibit expression or functionality of said protein encodedby said polynucleotide in said yeast cell.
 6. The method according toclaim 5, wherein step (B) comprises monitoring the level of said proteinproduced in said cell.
 7. The method according to claim 6, wherein step(B) comprises effecting a two-dimensional gel electrophoresis.
 8. Themethod according to claim 5, wherein step (B) comprises monitoring thelevel of m RNA encoded by said polynucleotide and produced by sais cell.9. The method according to claim 8, wherein step (B) comprises effectinga Northern blot, a primer extension, or a ribonuclease protection assay.10. The method according to claim 5, wherein step (B) comprisesmonitoring the level of kinase activity within said yeast cell, whereinsaid kinase activity typifies said protein.
 11. The method according toclaim 10, wherein step (B) comprises: (A) labeling ATP with ³²P invitro; (B) running cellular proteins on a polyacrylamide gel; and (C)determining the amount of ³²P labeled protein using autoradiography. 12.The method according to claim 5, wherein a promoter is operably linkedto a reporter gene and wherein step (B) comprises monitoring the levelof transcription of said reporter gene within said yeast cell.
 13. Themethod according to claim 12, wherein said reporter gene is a luciferasegene and luciferase activity is monitored using a luminometer.
 14. Anisolated polynuoleotide encoding the amino acid sequence of SEQ ID. NO.4.
 15. A culture of a bacterial strain containing the lambda phageλSG15.1.